Basic and translational research has investigated the role of nm23 in the regulation of tumor metastasis. Nine transfection studies have documented that overexpression of nm23 in various tumor cell lines resulted in a 50-90% decrease in tumor metastatic potential in vivo. The biochemical mechanism whereby Nm23 suppresses metastatic potential is under investigation. We have previously reported that a histidine dependent protein phosphotransferase activity correlated with Nm23-H1 suppression of tumor cell motility, an aspect of metastasis. Histidine protein kinases function in prokaryotic and lower eukaryotic organisms in the "two component" signal transduction system. Using a computer homology search, we have identified three mammalian proteins which share amino acid sequences with eukaryotic two-component response regulators. One of these proteins, the Kinase suppressor of ras (Ksr), is phosphorylated by Nm23-H1 in vitro in a histidine protein kinase reaction. Phosphoamino acid analysis suggests mostly serine phosphorylation of Ksr, and the sites are being identified. Ksr is thought to be a scaffold molecule in the Map kinase pathway. The data suggests the hypothesis that Nm23-H1 may function through modulation of signal transduction in this pathway. Translational research on nm23 proposes that elevation of Nm23 expression in micrometastatic or overtly metastatic breast or other carcinomas may limit colonization, motility and de-differentiation, with a clinical benefit. In order to determine how to elevate breast carcinoma Nm23 expression a model system consisting of four well characterized human cell lines differing in Nm23 expression and in vivo metastatic potential was used. The nm23-H1 promoter was cloned and a 2.1 kb fragment shown to confer differential expression when transfected into each cell line tethered to a reporter gene. Two observations have been made in this model system: (1)Deletion analysis revealed a 248 bp promoter fragment which contributed to high- vs. low Nm23-H1 expression. Within this fragment a striking pattern of transcription factor binding sites emerged: Sites known to determine mammary specific gene expression in the MMTV-LTR and milk genes (MAF, NF1, F11) were all present; deletion of the MAF and, to a lesser extent, NF1 sites reduced nm23-H1 directed reporter expression. Future research will investigate those factors binding the MAF and NF1 sites for their effects on Nm23-H1 expression. (2) One metastatically competent breast carcinoma cell line, MDA-MB-231, exhibited altered DNA methylation of the nm23-H1 promoter on southern blots. Studies using 5-aza-2'-deoxycytidine (5azadC), a DNA methylation inhibitor, increased Nm23 expression in this cell line, and three others, at a site more 5' from those identified in (1). In collaboration with Baylor University, we are asking whether differences in the DNA methylation status of the nm23-H1 promoter are found in human breast carcinomas. In vivo investigation of 5azadC is underway in mice.